The goal of this research is 3-fold: 1. Examine the mode of interaction between the protein units of the Beta-adrenoceptor sensitive adenylate cyclase: (a) Define the subunit composition of the adenylate cyclase effector system. This task will be approached by solubilization and purification of the adenylate cyclase in the detergent lauryl-sucrose (LS) in which all components are active. The components of the Beta-adrenoceptor dependent adenylate cyclase in its GDP (or GDPBetaS) basal state and its GppNHp active state will be examined using anti-GSAlpha antibodies raised against unique sequences and anti-GBeta antibodies. We possess anti-Beta-receptor and anti-GBeta antibodies, and are in the process of preparing anti-GSAlpha and anti-GiAlpha antibodies against unique sequences. We hope to determine the stoichiometry of the subunits GSAlpha:GBeta:C in the complexes. This information is crucial for understanding the mechanism of adenylate cyclase regulation by GS and Gi in view of our data which suggest a mechanism different from the popular "dissociation G-model". (b) We hope to be able to examine whether Gi interacts directly with C or with GS in the complex by examining the whereabouts of Gi during purification of the GS-cyclase complexes in LS. (c) Examine the Beta1-adrenoceptor to GS interaction and identify the GS subunits that interact with the receptor. Receptor and GS will be co-reconstituted, cross-linked, subjected to SDS-PAGE and analyzed by specific antibodies to receptor, GSAlpha and GBeta. The effect of receptor and GS specific ligands on the GS/R interaction will be examined. 2. To follow up our preliminary studies on the phospholipid specificity of R to GS to C coupling. We propose to study R to GS and R to GS to C coupling as a function of phospholipid composition in the reconstituted system. Since thee reconstituted system is more sensitive to phospholipid composition when GTP is used, we shall focus on the use of GTP. We shall try to correlate the kinetic parameters of the reconstituted system with: (i) the chemistry of the phospholipids and other additives used for reconstitution and (ii) the physical parameters of the reconstituted bilayer. This will allow us to discriminate between the role of the physical state of the membrane and that of specific lipid components on the transmembrane signaling. 3. Define the role of glycolipids, if any, on the coupling between the Beta-adrenoceptor and the components of adenylate cyclase. In parallel, we intend to pursue our search for the identity of the glycolipid which is tightly associated with the turkey erythrocyte Beta-adrenoceptor and, when found, examine its role in the interaction between the receptor and adenylate cyclase.